The present invention relates generally to methods for detecting fungi and, more particularly, to methods for the detection and quantitation of the fungus Stachybotrys chartarum by means of genetic amplification of a specimen.
Molds are ubiquitous in nature and are essential in nutrient cycling. The habitat or habitats that a mold occupies depend on several factors such as the kind and availability of nutrients, competition and spore dispersal. Fungi can occupy natural and man-made habitats in indoor and outdoor environments. These habitats include dead or living plants, decaying or freshly cut wood, food, grains, water and soil. Man-made products such as paint, wallpaper, and cellulose products (e.g., paper, cardboard, and wood derivatives) can be colonized and damaged by fungi, especially under humid or wet conditions. Certain molds can produce toxins that can cause health effects upon direct contact with skin, inhalation or ingestion.
Traditional methods of fungal identification include culture and microscopy analyses. However, these methods are laborious, time-consuming and require expertise. In addition, certain fungi are capable of causing health effects whether they are culturable or non-culturable. Other fungi are unable to produce classical structures under laboratory conditions that are necessary for identification. Stachybotrys chartarum is a toxigenic mold that has been implicated in the appearance of health effects in exposed individuals. This slow growing mold can colonize wet materials composed of cellulose. However, due to its specific nutrient and humidity requirements and the competition of other fungi, S. chartarum is often underestimated in traditional culture analyses.
Recently, analytical methods have been developed for rapidly and accurately detecting airborne bacteria (Alvarez, A. J., Buttner, M. P., Toranzos, G. A. et al. (1994). The use of solid-phase polymerase chain reaction for the enhanced detection of airborne microorganisms. Applied and Environmental Microbiology 60, 374-376; Alvarez, A. J., Buttner, M. P. and Stetzenbach, L. D. (1995). PCR for bioaerosol monitoring: sensitivity and environmental interference. Applied and Environmental Microbiology 61, 3639-3644), virus (Sawyer, M. H., Chamberlin, C. J., Wu, Y. N., Aintablian, N., and Wallace, M. R. (1994). Detection of varicella-zoster virus DNA in air samples from hospital rooms. Journal of Infectious Disease 169, 91-94) and fungi (Haugland, R. A., Vesper, S. J. and Wymer, L. J. (1999). Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with the TaqMan(trademark) fluorogenic probe system. Molecular and Cellular Probes 13, 329-340; Leenders, A.C.A.P., Van Belkum, A., Behrendt, M., Luijendijk, A. and Verbrugh, H. A. (1999). Density and molecular epidemiology of Aspergillus in air and relationship to outbreaks of Aspergillus infection. Journal of Clinical Microbiology 37, 1752-1757; Vesper, S., Dearborn, D. G., Yike, I. et al. (2000). Evaluation of Stachybotrys chartarum in the house of an infant with pulmonary hemorrhage: quantitative assessment before, during, and after remediation. Journal of Urban Health 77, 68-84). These methods use the polymerase chain reaction (PCR) to detect specific microorganisms by amplifying DNA sequences unique to the organism of interest. To use the PCR technique, sequence information must be first identified for a specific target DNA segment. Once an appropriate DNA sequence has been identified, oligonucleotide primers are selected, synthesized, and then tested for sensitivity, specificity, and selectivity. A fluorogenic nuclease assay in conjunction with a sequence detector (ABI PRISM 7700 Sequence Detection System, Applied Biosystems, Foster City, Calif.) has recently been developed as a means to amplify and quantitate PCR products, thus, eliminating the need for post-PCR gel electrophoresis for visualization of results (Heid, C. A., Stevens, J., Livak, K. J. and Williams, P. M. (1996). Real time quantitative PCR. Genome Research 6, 986-994). This method utilizes a fluorescently labeled oligonucleotide probe that anneals between the primers of choice as the amplification reaction proceeds, allowing for the determination of starting copy number of target DNA. The TaqMan(trademark) assay that is integral to this quantitative technology has been previously validated by other researchers with DNA extracted from Mycobacterium tuberculosis, Listeria monocytogenes and Salmonella.
PCR detection of S. chartarum has been reported (Haugland, R. A. and Heckman, J. L. (1998). Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum. Molecular and Cellular Probes 12, 387-396; Haugland et al., 1999; Vesper et al., 2000), and quantitative PCR (QPCR) with the TaqMan(trademark) assay has been used for the detection of S. chartarum in pure culture and air samples. However, quantitation of the target organism was estimated based on the co-amplification of another fungus (i.e., Geotrichum candidum) and not on direct comparison to S. chartarum standards (absolute quantitation). The method of estimated quantitation requires that the organisms co-amplifying have identical primer binding sites and amplification efficiencies, requiring the need for post-PCR processing in order to distinguish the products generated (Heid et al., 1996). In addition, estimated quantitation is inaccurate in cases where PCR inhibitors co-extract with the DNA (Desjardin, L. E., Chen, Y., Perkins, M. D., Teixeira, L., Cave, M. D. and Eisenach, K. D. (1998). Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis. Journal of Clinical Microbiology 36, 1964-1968; Haugland et al., 1999).
Therefore, there exists a need for the development of QPCR methods for the detection and absolute quantitation of S. chartarum. 
Methods consistent with the present invention address this need and others by employing QPCR with novel primers for detecting and quantitating S. chartarum without the necessity of further employing estimated quantitation techniques. Quantitation of samples suspected of containing S. chartarum, consistent with the present invention, may be based on direct comparison to S. chartarum standards (absolute quantitation), thus, avoiding the inaccuracies of estimated quantitation where PCR inhibitors may co-extract with the DNA. The primer and probe set used in QPCR consistent with the present invention may include oligonucleotide primers and a fluorescent probe that were designed from the internal transcribed spacer region (ITS 1) of the 18S rRNA gene of the species S. chartarum. 
In accordance with the purpose of the invention as embodied and broadly described herein, a method for detecting the fungus Stachybotrys chartarum includes isolating DNA from a sample suspected of containing the fungus Stachybotrys chartarum; subjecting the DNA to polymerase chain reaction amplification utilizing at least one primer, wherein the at least one primer comprises one of (SEQ ID NO: 1) 5xe2x80x2GTTGCTTCGGCGGGAAC3xe2x80x2(SEQ ID NO: 2) 5xe2x80x2TTTGCGTTTGCCACTCAGAG3xe2x80x2, (SEQ ID NO: 3) 5xe2x80x2ACCTATCGTTGCTTCGGCG3xe2x80x2, and (SEQ ID NO: 4) 5xe2x80x2GCGTTTGCCACTCAGAGAATACT3xe2x80x2base sequence; and detecting the fungus Stachybotrys chartarum by visualizing the product of the polymerase chain reaction.
In another exemplary embodiment consistent with the invention, a primer set for detecting Stachybotrys chartarum using polymerase chain reaction includes a first primer comprising a base sequence (SEQ ID NO: 1) 5xe2x80x2GTTGCTTCGGCGGGAAC3xe2x80x2; and a second primer comprising a base sequence (SEQ ID NO: 2) 5xe2x80x2TTTGCGTTTGCCACTCAGAG3xe2x80x2.
In a further exemplary embodiment consistent with the invention, a primer set for detecting Stachybotrys chartarum using polymerase chain reaction includes a first primer comprising a first base sequence (SEQ ID NO: 3) 5xe2x80x2ACCTATCGTTGCTTCGGCG3xe2x80x2; and a second primer comprising a second base sequence (SEQ ID NO: 4) 5xe2x80x2GCGTTTGCCACTCAGAGAATACT3xe2x80x2.
In an additional exemplary embodiment consistent with the invention, a primer and probe set for detecting the fungus Stachybotrys chartarum using polymerase chain reaction includes a forward primer comprising base sequence (SEQ ID NO: 1) 5xe2x80x2GTTGCTTCGGCGGGAAC3xe2x80x2; a reverse primer comprising a base sequence (SEQ ID NO: 2) 5xe2x80x2TTTGCGTTTGCCACTCAGAG3xe2x80x2; and a probe comprising a base sequence (SEQ ID NO: 5) 6-FAM-5xe2x80x2CTGCGCCCGGATCCAGGC3xe2x80x2-TAMRA.
In another exemplary embodiment consistentwith the invention, a primer and probe set for detecting the fungus Stachybotrys chartarum using polymerase chain reaction, includes a forward primer comprising a first base sequence (SEQ ID NO: 3) 5xe2x80x2ACCTATCGTTGCTTCGGCG3xe2x80x2; a reverse primer comprising a second base sequence (SEQ ID NO: 4) 5xe2x80x2GCGTTTGCCACTCAGAGAATACT3xe2x80x2; and a probe comprising a base sequence (SEQ ID NO: 5) 6-FAM-5 xe2x80x2CTGCGCCCGGATCCAGGC3xe2x80x2-TAMRA.
In a further exemplary embodiment consistent with the invention, a method for detecting the presence of the fungus Stachybotrys chartarum includes obtaining a sample from the environment; extracting DNA from the sample; and amplifying the extracted DNA by polymerase chain reaction utilizing one or more primers to obtain an indication of the presence of Stachybotrys chartarum in the sample, wherein the one or more primers comprise at least one of a (SEQ ID NO: 1) 5xe2x80x2GTTGCTTCGGCGGGAAC3, (SEQ ID NO: 2) 5xe2x80x2TTTGCGTTTGCCACTCAGAG3xe2x80x2, (SEQ ID NO: 3) 5xe2x80x2ACCTATCGTTGCGGCG3xe2x80x2, and (SEO ID NO: 4) 5xe2x80x2GCGTTTGCCACTCAGAGAATAcT3xe2x80x2base sequence.
In yet another cxemplary embodiment consistent with the present invention, a method for detecting the presence of the fungus Stachybotrys chartarum includes obtaining a sample from the environment; extracting DNA from the sample; and amplifying the extracted DNA by polymerase chain reaction utilizing a primer set to obtain an indication of the presence of Stachybotrys chartarum in the sample, wherein the primer set comprises: a forward primer comprising a first base sequence (SEQ ID NO: 3) 5xe2x80x2ACCTATCGTTGCTTCGGCG3xe2x80x2, and a reverse primer comprising a second base sequence (SEQ ID NO: 4) 5xe2x80x2GCGTTTGCCACTCAGAGAATACT3xe2x80x2.
In a further exemplary embodiment consistent with the invention, a method for identifying and quantifying the presence of the fungus Stachybotrys chartarum in a collected sample includes obtaining a primer set and probe that is specific for the fungal species Stachybotrys chartarum; collecting the sample from the environment; extracting the sample""s DNA; obtaining DNA standards from a culture of Stachybotrys chartarum; determining the concentration of Stachybotrys chartarum spores in the DNA standards; amplifying by polymerase chain reaction each of the DNA standards and the collected sample""s DNA using the obtained primer set and probe; and comparing amplification plots obtained by polymerase chain reaction of each of the DNA standards and the collected sample""s DNA to obtain an indication of the presence of the fungus Stachybotrys chartarum in the collected sample and a concentration of the fungus Stachybotrys chartarum in the collected sample.